The mechanism of intracellular sorting of prohormones, pro-opiomelanocortin (POMC, pro-ACTH/endorphin) pro-enkephalin (pro-ENK) and pro-insulin to the regulated secretory pathway (RSP) was investigated. These prohormones undergo homotypic oligomerization, as a concentration step as they traverse the cell from the site of synthesis in the endoplasmic reticulum to the trans-Golgi network(TGN)where they are sorted into dense-core granules of the regulated secretory pathway for processing and secretion. Site-directed mutagenesis studies identified a concensus sorting motif consisting of two acidic residues, 12-15 Angs apart from each other, exposed on the surface of these molecules, and two hydrophobic residues, 5-7 Angs away from the acidic residues which are necessary for sorting to the RSP. For POMC, the residues are D10, L11; E14, L18 located at the N-terminus. A similar sorting motif consisting of residues D18, I19; E29,L32 was found in the N-terminus of pro-enkephalin. In monomeric proinsulin, the sorting signal motif consists of residues E13 and L17 located on the B chain and L16 and E17 located on the A chain. In hexameric proinsulin, residue E13 on the B chain is buried and the motif is contributed by the two residues in the A chain from two adjacent proinsulin dimers in the hexamer. A RSP sorting receptor that was specific for the sorting signal of POMC, pro-insulin and pro-enkephalin was identified as membrane carboxypeptidase E (CPE). The two acidic residues in the prohormone sorting motif specifically interact with two basic residues, R255 and K260, of the sorting receptor, carboxypeptidase E (CPE), to effect sorting to the RSP. We showed that CPE is a transmembrane protein which is anchored via its C-terminal amphipathic tail to unique cholesterol-glycosphingolipid rich microdomains known as rafts, in the TGN. Removal of cholesterol from secretory granule membranes by methyl beta-cyclodextrin resulted in the inability of CPE to bind cargo; and cholesterol depletion by treatment of cells with lovastatin resulted in lack of sorting of CPE and POMC to the RSP. Thus membrane association is essential for the prohormone sorting receptor function of CPE at the TGN, as well as its own sorting to the RSP. Depletion of CPE by antisense RNA in Neuro2a cells caused missorting of prohormones to the constitutive pathway, indicating that CPE functions as a sorting receptor in vivo. Using a mouse model which synthesizes a mutant CPE that is differentially degraded in pituitary and pancreas, we were able to show a correlation between lowered CPE levels and the degree of missorting of endogenous prohormones in the cells of these tissues. These studies provide evidence for a sorting signal/receptor mediated mechanism for targeting prohormones to the regulated secretory pathway in neuro-endocrine cells. The intracellular sorting of genetically mutated proinsulins found in hyperproinsulinemia patients who have abnormally high levels of plasma proinsulin was investigated to understand the molecular basis of these forms of diabetes. One form of mutant proinsulin found in these patients, HisB10Asp, which is unable to hexamerize but forms dimers, was found to be missorted to the constitutive pathway and secreted in an unregulated manner when transfected into a cell line. Molecular modelling of the dimer of this mutant proinsulin predicted that the molecular distance of the two acidic residues of the RSP sorting signal motif would be too large to allow interaction with the basic residues in the binding site of the sorting receptor, CPE. Indeed in vitro binding studies showed that this mutant did not bind to CPE, thus resulting in its inability to be sorted to the RSP for processing to insulin and secretion in a secretogogue-dependant manner. Other hyperproinsulinemia proinsulin mutants, Arg65Pro and Arg65Leu were also found to be secreted constitutively and not stored. The high levels of secreted mutant proinsulins in the plasma of these patients are therefore due to defects in sorting, processing, storage and secretion of these molecules resulting from their genetic structural alterations. Formation of large dense-core granules(LDCG) at the TGN is essential for regulated secretion of hormones and neuropeptides from neuroendocrine cells. Our recent studies uncovered a master on/off switch , chromogranin A (CgA), that controls the formation of LDCG in neuroendocrine cells. Depletion of CgA in rat PC12 cells using antisense technology resulted in the loss of LDCG, regulated secretion and degradation of granule proteins including CgB and synaptotagmin. Overexpression of bovine CgA in these cells rescued the wild type phenotype. In a mutant endocrine cell line lacking CgA, LDCGs and regulated hormone secretion, transfection of CgA restored the wild type phenotype in these cells. Thus CgA serves an important higher-order physiological role in controlling hormone secretion through regulating secretory granule biogenesis in endocrine and neuroendocrine cells.